G protein coupled receptors (GPCRs) that activate cytoplasmic trimeric G proteins play critical roles in diverse cellular signaling pathways. However, the mechanisms by which ligand binding to receptors effect changes in the G proteins are not understood. This proposal focuses on the alpha-factor receptor, encoded by the yeast STE2 gene as a model for studying G protein activation. The role of this receptor in yeast mating has led to development of sensitive screens and selections both for and against receptor function that allow isolation of receptors with altered function from random mutational libraries. Since the STE2 gene product is similar to, and functionally interchangeable with, other GPCRs, the results and techniques used should be widely applicable. The project has four major aims: I. Identification of interacting surfaces and relative motions of transmembrane segments. The three-dimensional arrangement of the seven predicted transmembrane helices of GPCRs is not known. By combining the isolation of loss-of function and second-site intragenic suppressor mutations with intramolecular disulfide crosslinking, interactions between transmembrane segments will be identified. These interactions will be used as constraints in structural modeling of the alpha-factor receptor. In addition, effects of intramolecular crosslinking on receptor function will be used to detect activation-dependent motions of transmembrane segments. II. Localization of the site of ligand binding. This will be accomplished by screening for receptor mutations that interfere with ligand binding and for receptor mutations that can compensate for alterations in the structure of the ligand. III. Determination of the mechanism of interactions between receptors. Our laboratory has isolated dominant negative mutant receptors that interfere with the function of co-expressed normal receptors. In addition, signaling by hypersensitive and constitutively active mutant receptors can be suppressed by co-expressed normal receptors. Such inter-receptor interactions could result from oligomerization of receptors during signaling or sequestration of G protein by inactive receptors. These possibilities will be distinguished by crosslinking of putative oligomers, by examining the effects of G protein overexpression, and by covalently fusing the G protein alpha subunit to various receptor alleles. IV. Identification of sites of receptor-G protein interactions. To identify particular amino acid residues at sites of contact between the receptor and the G protein, genetic screens will be conducted for G protein mutations that specifically block interactions with the receptor and for receptor mutations that suppress G protein mutations.